The effect of increasing risk and challenge in the school playground on physical activity and weight in children: a cluster randomised controlled trial (PLAY).

BACKGROUND: To investigate whether changing the play environment in primary schools to one that includes greater risk and challenge increases physical activity and reduces body mass index (BMI). SUBJECTS/METHODS: A 2-year cluster randomised controlled trial was undertaken in 16 New Zealand schools (years 1-8). Intervention schools (n=8) redesigned their play environments to encourage imaginative and independent free play by increasing opportunities for risk and challenge (for example, rough-and-tumble play), reducing rules and adding new playground components (for example, loose parts). Control schools (n=8) were asked to not change their play environment. A qualified playworker rated all school play environments at baseline and 1 year. Primary outcomes were moderate-to-vigorous physical activity (7-day accelerometry) and BMI z-score, collected in 840 children at baseline, 1 and 2 years. Data were analysed using generalised estimating equations. RESULTS: Multiple changes were made to the school play environments resulting in a significant difference in overall play evaluation score between intervention and control schools of 4.50 (95% confidence interval: 1.82 to 7.18, P=0.005), which represents a substantial improvement from baseline values of 19.0 (s.d. 3.2). Overall, schools liked the intervention and reported many benefits, including increased physical activity. However, these beliefs did not translate into significant differences in objectively measured physical activity, either as counts per minute (for example, 35 (-51 to 120) during lunch break) or as minutes of moderate-to-vigorous physical activity (0.4, -1.1 to 2.0). Similarly, no significant differences were observed for BMI, BMI z-score or waist circumference at 1 or 2 years (all P>0.321). CONCLUSIONS: Altering the school play environment to one that promoted greater risk and challenge for children did not increase physical activity, nor subsequently alter body weight. Although schools embraced the concept of adding risk and challenge in the playground, our findings suggest that children may have been involved in different, rather than additional activities.

Molecular determinants of state-dependent block of Na+ channels by local anesthetics.

Sodium ion (Na+) channels, which initiate the action potential in electrically excitable cells, are the molecular targets of local anesthetic drugs. Site-directed mutations in transmembrane segment S6 of domain IV of the Na+ channel alpha subunit from rat brain selectively modified drug binding to resting or to open and inactivated channels when expressed in Xenopus oocytes. Mutation F1764A, near the middle of this segment, decreased the affinity of open and inactivated channels to 1 percent of the wild-type value, resulting in almost complete abolition of both the use-dependence and voltage-dependence of drug block, whereas mutation N1769A increased the affinity of the resting channel 15-fold. Mutation I1760A created an access pathway for drug molecules to reach the receptor site from the extracellular side. The results define the location of the local anesthetic receptor site in the pore of the Na+ channel and identify molecular determinants of the state-dependent binding of local anesthetics.

Distribution of serotonergic neurons and processes in the lamprey spinal cord.

The distribution of serotonin in the spinal cord in two species of lamprey, Ichthyomyzon unicuspis and Petromyzon marinus, was studied by indirect immunofluorescence techniques. Multipolar cell bodies containing serotonin-like immunoreactivity were found along the length of the spinal cord, along the midline and slightly ventral to the central canal. These cell bodies send a diffuse projection of processes throughout the spinal cord, including: (1) a dense projection to the ventral surface; (2) a strong projection to the ventromedial longitudinal fiber tracts; (3) a less intense projection to the dorsal longitudinal fiber tracts; and (4) a weak projection to the lateral fiber tracts. Lesion experiments showed that processes descending from the brain or rostral spinal cord provide a major projection to the lateral fiber tracts and smaller contributions to the dorsal and ventromedial fiber tracts. Fluorescent processes were also observed in the dorsal roots and serotonergic peripheral cell bodies were seen adjacent to the dorsal roots. Our results suggest that the serotonergic innervation of the lamprey spinal cord arises from three sources: spinal interneurons, descending tracts and peripheral (possibly sensory) input. This provides an anatomical substrate for our recent finding that serotonin modulates the central pattern generator for locomotion in the lamprey spinal cord.

Physiology, medicine, long-duration space flight and the NSBRI.

The hazards of long-duration space flight are real and unacceptable. In order for humans to participate effectively in long-duration orbital missions or continue the exploration of space, we must first secure the health of the astronaut and the success of such missions by assessing in detail the biomedical risks of space flight and developing countermeasures to these hazards. Acquiring the understanding necessary for building a sound foundation for countermeasure development requires an integrated approach to research in physiology and medicine and a level of cooperative action uncommon in the biomedical sciences. The research program of the National Space Biomedical Research Institute (NSBRI) was designed to accomplish just such an integrated research goal, ameliorating or eliminating the biomedical risks of long-duration space flight and enabling safe and productive exploration of space. The fruits of these labors are not limited to the space program. We can also use the gained understanding of the effects and mechanisms of the physiological changes engendered in space and the applied preventive and rehabilitative methods developed to combat these changes to the benefit of those on Earth who are facing similar physiological and psychological difficulties. This paper will discuss the innovative approach the NSBRI has taken to integrated research management and will present some of the successes of this approach.

A mutation in segment IVS6 disrupts fast inactivation of sodium channels.

Na(+)-channel inactivation is proposed to occur by binding of an intracellular inactivation gate to a hydrophobic inactivation gate receptor in the intracellular mouth of the pore. Amino acid residues in transmembrane segment S6 of domain IV (IVS6) that are critical for fast inactivation were identified by alanine-scanning mutagenesis. Mutant VIL1774-6AAA, in which three adjacent residues (Val-Ile-Leu) at the intracellular end of segment IVS6 were converted to alanine, had substantial (> 85%) sustained Na+ currents remaining 15 ms after depolarization, while a nearby mutation of three residues to alanine had no effect. Single-channel analysis revealed continued reopenings late in 40-ms depolarizing pulses indicating that inactivation was substantially impaired compared to wild type. The mean open time for VIL1774-6AAA was longer than wild type, suggesting that this mutation also decreases the rate of entry into the fast inactivated state. These results suggest that residues near the intracellular end of segment IVS6 are critical for fast Na(+)-channel inactivation and may form part of the hydrophobic receptor site for the fast inactivation gate.

A critical role for transmembrane segment IVS6 of the sodium channel alpha subunit in fast inactivation.

Fast Na+ channel inactivation is thought to occur by the binding of an intracellular inactivation gate to regions around or within the Na+ channel pore through hydrophobic interactions. Previous studies indicate that the intracellular loop between domains III and IV of the Na+ channel alpha subunit (LIII-IV) forms the inactivation gate. A three-residue hydrophobic motif (IFM) is an essential structural feature of the gate and may serve as an inactivation particle that binds within the pore. In this study, we used alanine-scanning mutagenesis to examine the functional role of amino acid residues in transmembrane segment IVS6 of the Na+ channel alpha subunit in fast inactivation. Mutant F1764A, in the center of IVS6, and mutant V1774A, near its intracellular end, exhibited substantial sustained Na+ currents at the end of 30-ms depolarizations. The double mutation F1764A/V1774A almost completely abolished fast inactivation, demonstrating a critical role for these amino acid residues in the process of inactivation. Single channel analysis of these three mutants revealed continued reopenings late in 40-ms depolarizing pulses, indicating that the stability of the inactivated state was substantially impaired compared with wild type. In addition, the cumulative first latency distribution for the V1774A mutation contained a new component arising from opening transitions from the destabilized inactivated state. Substitution of multiple amino acid residues showed that the disruption of inactivation was not correlated with the hydrophobicity of the substitution at position 1774, in contrast to the expectation if this residue interacts directly with the IFM motif. Thermodynamic cycle analysis of simultaneous mutations in the IFM motif and in IVS6 suggested that mutations in these two regions independently disrupt inactivation, consistent with the conclusion that they do not interact directly. Furthermore, a peptide containing the IFM motif (acetyl-KIFMK-amide) restored inactivation to the F1764A/V1774A IVS6 mutant, indicating that the binding site for the IFM motif remains intact in these mutants. These results suggest that the amino acid residues 1764 and 1774 in IVS6 do not directly interact with the IFM motif of the inactivation gate but instead play a novel role in fast inactivation of the Na+ channel.

Common molecular determinants of local anesthetic, antiarrhythmic, and anticonvulsant block of voltage-gated Na+ channels.

Voltage-gated Na+ channels are the molecular targets of local anesthetics, class I antiarrhythmic drugs, and some anticonvulsants. These chemically diverse drugs inhibit Na+ channels with complex voltage- and frequency-dependent properties that reflect preferential drug binding to open and inactivated channel states. The site-directed mutations F1764A and Y1771A in transmembrane segment IVS6 of type IIA Na+ channel alpha subunits dramatically reduce the affinity of inactivated channels for the local anesthetic etidocaine. In this study, we show that these mutations also greatly reduce the sensitivity of Na+ channels to state-dependent block by the class Ib antiarrhythmic drug lidocaine and the anticonvulsant phenytoin and, to a lesser extent, reduce the sensitivity to block by the class Ia and Ic antiarrhythmic drugs quinidine and flecainide. For lidocaine and phenytoin, which bind preferentially to inactivated Na+ channels, the mutation F1764A reduced the affinity for binding to the inactivated state 24.5-fold and 8.3-fold, respectively, while Y1771A had smaller effects. For quinidine and flecainide, which bind preferentially to the open Na+ channels, the mutations F1764A and Y1771A reduced the affinity for binding to the open state 2- to 3-fold. Thus, F1764 and Y1771 are common molecular determinants of state-dependent binding of diverse drugs including lidocaine, phenytoin, flecainide, and quinidine, suggesting that these drugs interact with a common receptor site. However, the different magnitude of the effects of these mutations on binding of the individual drugs indicates that they interact in an overlapping, but nonidentical, manner with a common receptor site. These results further define the contributions of F1764 and Y1771 to a complex drug receptor site in the pore of Na+ channels.

Evidence for a functional interaction between integrins and G protein-activated inward rectifier K+ channels.

Heteromultimeric G protein-activated inward rectifier K+ (GIRK) channels, abundant in heart and brain, help to determine the cellular membrane potential as well as the frequency and duration of electrical impulses. The sequence arginine-glycine-aspartate (RGD), located extracellularly between the first membrane-spanning region and the pore, is conserved among all identified GIRK subunits but is not found in the extracellular domain of any other cloned K+ channels. Many integrins, which, like channels, are integral membrane proteins, recognize this RGD sequence on other proteins, usually in the extracellular matrix. We therefore asked whether GIRK activity might be regulated by direct interaction with integrin. Here, we present evidence that mutation of the RGD site to RGE, particularly on the GIRK4 subunit, decreases or abolishes GIRK current. Furthermore, wild-type channels can be co-immunoprecipitated with integrin. The total cellular amount of expressed mutant GIRK channel protein is the same as the wild-type protein; however, the amount of mutant channel protein that localizes to the plasma membrane is decreased relative to wild-type, most likely accounting for the diminished GIRK current detected. GIRK channels appear to bind directly to integrin and to require this interaction for proper GIRK channel membrane localization and function.

A critical role for the S4-S5 intracellular loop in domain IV of the sodium channel alpha-subunit in fast inactivation.

Na+ channel fast inactivation is thought to involve the closure of an intracellular inactivation gate over the channel pore. Previous studies have implicated the intracellular loop connecting domains III and IV and a critical IFM motif within it as the inactivation gate, but amino acid residues at the intracellular mouth of the pore required for gate closure and binding have not been positively identified. The short intracellular loops connecting the S4 and S5 segments in each domain of the Na+ channel alpha-subunit are good candidates for this role in the Na+ channel inactivation process. In this study, we used scanning mutagenesis to examine the role of the IVS4-S5 region in fast inactivation. Mutations F1651A, near the middle of the loop, and L1660A and N1662A, near the COOH-terminal end, substantially disrupted Na+ channel fast inactivation. The mutant F1651A conducted Na+ currents that decayed very slowly, while L1660A and N1662A had large sustained Na+ currents at the end of 30-ms depolarizing pulses. Inactivation of macroscopic Na+ currents was nearly abolished by the N1662A mutation and the combination of the F1651A/L1660A mutations. Single channel analysis revealed frequent reopenings for all three mutants during 40-ms depolarizing pulses, indicating a substantial impairment of the stability of the inactivated state compared with wild type (WT). The F1651A and N1662A mutants also had increased mean open times relative to WT, indicating a slowed rate of entry into the inactivated state. In addition to these effects on inactivation of open Na+ channels, mutants F1651A, L1660A, and N1662A also impaired fast inactivation of closed Na+ channels, as assessed from measurements of the maximum open probability of single channels. The peptide KIFMK mimics the IFM motif of the inactivation gate and provides a test of the effect of mutations on the hydrophobic interaction of this motif with the inactivation gate receptor. KIFMK restores fast inactivation of open channels to the F1651A/L1660A mutant but does not restore fast inactivation of closed F1651A/L1660A channels, suggesting that these residues interact with the IFM motif during inactivation of closed channels. Our results implicate F1651, L1660, and N1662 of the IVS4-S5 loop in inactivation of both closed and open Na+ channels and suggest that the IFM motif of the inactivation gate interacts with F1651 and/or L1660 in the IVS4-S5 loop during inactivation of closed channels.

High affinity binding of pyrethroids to the alpha subunit of brain sodium channels.

Na+ channels are the primary molecular targets of the pyrethroid insecticides. Na+ channels consisting of only a type IIA alpha subunit expressed in Chinese hamster ovary cells responded to pyrethroid treatment in a normal manner: a sustained Na+ current was induced progressively after each depolarizing pulse in a train of stimuli, and this Na+ current decayed slowly on repolarization. These modified Na+ channels could be reactivated at much more negative membrane potentials (V0.5 = -139 mV) than unmodified Na+ channels (V0.5 = -28 mV). These results indicate that pyrethroids can modify the functional properties of the Na+ channel alpha subunit expressed alone by blocking their inactivation, shifting their voltage dependence of activation, and slowing their deactivation. To demonstrate directly the specific interaction of pyrethroids with the alpha subunit of voltage-gated Na+ channels, a radioactive photosensitive derivative, [3H]RU58487, was used in binding and photolabeling studies. In the presence of a low concentration of the nonionic detergent Triton X-100, specific pyrethroid binding to Na+ channels in rat brain membrane preparations could be measured and reached 75% of total binding under optimal conditions. Binding approached equilibrium within 1 hr at 4 degrees, dissociated with a half-time of approximately 10 min, and had K(D) values of approximately 58-300 nM for three representative pyrethroids. Specific pyrethroid binding was enhanced by approximately 40% in the presence of 100 nM alpha-scorpion toxin, but no allosteric enhancement was observed in the presence of toxins acting at other Na+ channel receptor sites. Extensive membrane washing increased specific binding to 89%. Photolabeling with [3H]RU58487 under these optimal binding conditions revealed a radiolabeled band with an apparent molecular mass of 240 kDa corresponding to the Na+ channel alpha subunit. Anti-peptide antibodies recognizing sequences within the alpha subunit were able to specifically immunoprecipitate the covalently modified channel. Together, these results demonstrate that the pyrethroids can modify the properties of cells expressing only the alpha subunit of Na+ channels and can bind specifically to a receptor site on the alpha subunit.